A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth.
Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor of this enzyme. This labeled vinyl ketone, [3H](Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.
Tasayco
M L
ML
Department of Chemistry, State University of New York, Stony Brook 11794-3400.
Prestwich
G D
GD
D000345:Affinity Labels; D000444:Aldehyde Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D042931:Aldehyde Oxidase; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D000466:Alkadienes / Q000138:chemical synthesis; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007915:Lepidoptera / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D009036:Moths / Q000201:enzymology*; D010084:Oxidation-Reduction; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014316:Tritium
D000345:Affinity Labels; D000447:Aldehydes; D000466:Alkadienes; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D014316:Tritium; C063060:1,11-hexadecadien-3-one; D000445:Aldehyde Oxidoreductases; D000444:Aldehyde Dehydrogenase; D042931:Aldehyde Oxidase